One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues both of plants and animals. One well known class of such peptides is the tachyplesins which were first isolated from the hemocytes of the horseshoe crab as described by Nakamura, T. et al. J Biol Chem (1988) 26:16709-16713. This article described the initial tachyplesin isolated from the Japanese species, Tachyplesin I, which is a 17-amino acid amidated peptide containing four cysteine residues providing two intramolecular cystine bonds. In a later article by this group, Miyata, T. et al. J Biochem (1989) 106:663-668, extends the studies to the American horseshoe crab and isolated a second tachyplesin, Tachyplesin II, consisting of 17 residues amidated at the C-terminus, also containing four cysteine residues and two intramolecular disulfide bonds. Two additional 18-mers, called polyphemusins, highly homologous to Tachyplesin II and containing the same positions for the four cysteine residues, were also isolated. Polyphemusin I and Polyphemusin II differ from each other only in the replacement of one arginine residue by a lysine. All of the peptides were described as having antifungal and antibacterial activity. A later article by Murakami, T. et al. Chemotherapy (1991) 37:327-334, describes the antiviral activity of the tachyplesins with respect to vesicular stomatus virus; Herpes Simplex Virus I & II, Adenovirus I, Reovirus II and Poliovirus I were resistant to inactivation by Tachyplesin I. Morimoto, M. et al. Chemotherapy (1991) 37:206-211, found that Tachyplesin was inhibitory to Human Immunodeficiency Virus. This anti-HIV activity was found also to be possessed by a synthetic analog of Polyphemusin II as described by Nakashima, H. et al. Antimicrobial Agents and Chemotherapy (1992) 1249-1255. Antiviral peptides have also been found in rabbit leukocytes as reported by Lehrer, R. I. et al. J Virol (1985) 54:467-472.
Another class of antimicrobial and antiviral peptides, the "protegrins" have been isolated from porcine leukocytes as reported by the present applicants in a paper by Kokryakov, V. N. et al. FEBS (1993) 337:231-236 An additional paper disclosing cationic peptides from porcine leukocytes was published by Mirgorodskaya, O. A. al. FEBS (1993) 330:339-342. Storici, P. et al. Biochem Biophys Res Comm (1993) 196:1363-1367, report the recovery of a DNA sequence which encodes a pig leukocyte antimicrobial peptide with a cathelin-like prosequence and is reported to be one of the protegrins.
Other important classes of cysteine-containing antimicrobial peptides include the defensins, .beta.-defensins and insect defensins. The defensins are somewhat longer peptides characterized by six invariant cysteines and three intramolecular cystine disulfide bonds. Defensins were described by Lehrer, R. I. et al. Cell (1991) 64:229-230; Lehrer, R. I. et al. Ann Rev Immunol (1993) 11:105-128. A review of mammalian-derived defensins by Lehrer, R. I. et al. is found in Annual Review Immunol (1993) 11:105-128; three patents have issued on the defensins: U.S. Pat. No. 4,705,777; U.S. Pat. No. 4,659,692; and U.S. Pat. No. 4,543,252. Defensins have been found in the polymorphonucleated leukocytes (PMN) of humans and of several other animals, as well as in rabbit pulmonary alveolar macrophages, and in murine small intestinal epithelial (Paneth) cells and in corresponding cells in humans.
.beta.-Defensins are found in bovine leukocytes and respiratory epithelial cells. See Selsted, M. E. et al. J Biol Chem (1993) 288:6641-6648, Tang, Y-Q. J Biol Chem (1993) 268:6649-6653, and Diamond, G. et al. Proc Natl Acad Sci (USA) (1991) 88:3952-3958, Diamond, G. et al. Proc Natl Acad Sci USA (1993) 90:4596-4600. Insect defensins have been reported by Lambert, J. et al. Proc Natl Acad Sci (USA) (1989) 88:262-265.
Antifungal and antibacterial peptides and proteins have also been found in plants (Broekaert, W. F. et. al. Biochemistry (1992) 31:4308-4314) as reviewed by Cornelissen, B. J. C. et al. Plant Physiol (1993) 101:709-712. Expression systems for the production of such peptides have been used to transform plants to protect the plants against such infection as described, for example, by Haln, R. et al. Nature (1993) 361:153-156.
Another group of antibacterial peptides which are isolated from avian leukocytes are the gallinacins. These peptides have cysteine distribution patters which are similar to the .beta.-defensins obtained from mammalian species. These peptides are described by Harwig, S. S. L. et al. in Techniques in Protein Chemistry V (1994) Academic Press Inc., New York, pages 81-88.
An interesting feature of many of the apparently unrelated naturally occurring antimicrobial peptides is that they are derived from precursors which have similar 5' regions highly homologous to a porcine cysteine proteinase inhibitor, cathelin. For example, Storici, P. et al. Biochem Biophys Res Commun (1993) 196:1363-1368, point out that the .beta.-defensins, as well as certain other classes of antimicrobial peptides share this common upstream region. These peptides include Bac5 (Gennaro, R. et al. Infect Immun (1989) 57:3142-3146 for which the cDNA was reported by Zanetti, M. et al. J Biol Chem (1993) 268:522-526); indolicidin (Selsted, M. E. et al. J Biol Chem (1992) 267:4292-4295; Delsal, G. et al. Biochem Biophys Res Commun (1992) 187:467-472); an acyclic dodecapeptide from bovine sources (Romeo, D. et al. J Biol Chem (1988) 263:9573-9575) for which a cDNA clone was reported by Storici, P., et al. FEBS (1992) 314:187-190); and CAP18 from rabbit leukocytes (Larrick, J. W. et al. Biochem Biophys Res Commun (1991) 179:170-175). This upstream sequence is also shared by the antibacterial 15 kD proteins, "p15", from rabbit polymorphonuclear leukocytes described by Levy, O. et al. J. Biol Chem (1993) 268:6058-6063. Still another member of this family is the PR39 peptide described by Storici, P. et al. Biochem Biophys Res Commun (1993) 196:1058-1065. All of these are characterized by prosequences homologous to the cysteine proteinase inhibitor, cathelin/PLCPI.
Taking advantage of this precursor homology, Pungercar, J. et al. FABS (1993) 336:284-288 (the December issue) utilized a PCR-derived probe obtained by amplification of the propeptide region of a rabbit Cap18 cDNA to screen a cDNA library prepared in .lambda.-GT11 from pig bone marrow. The retrieved cDNA clone contained a deduced amino acid sequence with the cathelin N-terminal sequence and a putative 100-amino acid residue mature protein rich in praline. These authors further suggest that the C-terminal 3-amino acid residue may be cleaved in the mature form to yield a 97-amino acid mature protein with multiple praline residues.
The effect of proline-rich regions on the structure and function of various proteins has been reviewed by Williamson, M. P. et al. Biochem J (1994) 297:249-260. Most of such proteins serve a structural or binding purpose since the presence of multiple praline residues imposes severe conformational constraints.
The present invention is directed to a family of proline-rich antimicrobial peptides containing about 79 amino acid residues with repeating decamer regions. The invention peptides are particularly effective against Gram-negative bacteria and also are capable of binding lipopolysaccharide (LPS) thus having the ability to mitigate the symptoms of septic shock. They are useful as preservatives and therapeutics. This class of peptides is designated "prophenins".